Construction of Interlayer Coupling Diatomic Nanozyme with Peroxidase‐Like and Photothermal Activities for Efficient Synergistic Antibacteria

Abstract Pathogenic bacteria are the main cause of bacterial infectious diseases, which have posed a grave threat to public health. Single‐atom nanozymes have emerged as promising candidates for antibacterial applications, but their activities need to be further improved. Considering diatomic nanozymes exhibit superior metal loading capacities and enhanced catalytic performance, a new interlayer coupling diatomic nanozyme (IC‐DAN) is constructed by modulating the coordination environment in an atomic‐level engineering. It is well demonstrated that IC‐DAN exhibited superior peroxidase‐mimetic activity in the presence of H2O2 to yield abundant ∙OH and possessed high photothermal conversion ability, which synergistically achieves efficient antibacterial therapy. Therefore, IC‐DAN shows great potential used as antibacterial agent in clinic and this study open a new route to developing high‐performance artificial enzymes.

Renishaw, China) with a 633 nm laser source.The Fe K-edge X-ray absorption nearedge structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data were collected on the 06ID-1 Hard X-ray Micro Analysis (HXMA) beamline at the Canadian Light Source.

/(Km+[S])
Where A represents the absorbance values, k represents the molar extinction coefficient.
In this case, k = 39000 M cm -1 .c refers to the concentration of the chromogenic substances while t refers to time.
In this equation, V0 is the conversion rate, Vmax is the maximal rate of conversion rate, Κm is the Michaelis constant, and [S] is the substrate concentration.The Κm is equal to the substrate concentration at which the conversion rate is half of Vmax.Km means the affinity of the enzyme for the substrate namely a lesser Km value implies a better affinity.

Calculation Methods
The spin-polarized density functional theories (DFT) were carried out by using the Vienna Ab initio Simulation Package (VASP) [1] .The Perdew-Burke-Ernzerhof generalized-gradient approximation functional was used to describe the interaction between electrons [2] .The energy cutoff was set to 400 eV.The 2×2×1 Gamma-Centered k-points grid was set during the calculations.The vacuum region was set to be 15 Å in z direction to prevent the interaction between two adjacent surfaces.The energy convergence was set to 10 -5 eV.The convergence criteria of the forces were selected as 0.02 eV Å −1 .
The reaction Gibbs free energy (ΔG) is defined as ΔG = ΔE + ΔZPE -TΔS, ΔE is the reaction energy, ΔZPE is zero-point energies, T is the temperature (298.15K), ΔS is the difference in entropy from vibrational frequency calculations.The entropies of gas phase H2O2, H2 and H2O are obtained from the NIST database with standard condition [3] .

Biological Experiments
Instruments.Fluorescence imaging by using an inverted confocal fluorescence microscope (FV3000, Olympus).808 nm near-infrared (NIR) laser was bought from Jiuyi Laser Technology (Beijing, China).The thermal imaging of IC-DAN solution and mice was imaged by an infrared thermal camera (HIKVISION, China).Absorbance was measured by a microplate reader (Cytation5, BioTek).Measurement of Peroxidase-Like Activity.The peroxidase (POD)-like activity of IC-DAN was measured using TMB as a probe in the HAc-NaAc buffer.The absorbance at 652 nm was recorded using a Cytation 5 microplate reader.To study the effect of pH on POD-like activity of IC-DAN, we added IC-DAN (20 μg mL -1 ) and 800 μM TMB solution into HAc-NaAc buffer (pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), respectively.The mixture was incubated for 10 min and the final 652 nm absorbance of the mixture was detected by a microplate reader.To further measured the kinetic assays of IC-DAN, the kinetic assays of IC-DAN (20 μg mL -1 ) with TMB as the substrate were performed by adding 100 μM H2O2 and different concentrations of TMB solution (0, 200, 400, 800, 1000 μM) in a 0.01 M HAc-NaAc buffer (pH = 4).The kinetic assays of IC-DAN (20 μg mL -1 ) with H2O2 as the substrate were performed by adding 800 μM TMB and different concentrations of H2O2 solution (0, 10, 25, 50, 100 μM) in a HAc-NaAc buffer (pH = 4).To measure the effect of temperature on the POD-like activity of IC-DAN.The kinetic assay was conducted at different temperatures (20, 30, 40, 50, and 60°C) in a 0.01 M HAc-NaAc buffer (pH = 4) with IC-DAN (10 μg mL - Photothermal Effect of IC-DAN.IC-DAN was dispersed in ultra-pure water at three concentrations (10, 20, or 30 μg mL -1 ).Then 100 μL solution was added to a centrifuge tube and irradiated with an 808 nm NIR laser source at different power densities (0.6, 0.8, or 1.0 W cm -2 ) for 5 min.The temperatures and photographs of the NIR irradiated IC-DAN solution were recorded at 10 s intervals using an infrared imaging device.

Antibacterial Activity of IC-DAN. Bacterial culture and antibacterial activities in vitro
To assess the antibacterial effect of IC-DAN, five groups were established.
Bacteria without any treatment served as the control group named G1 and the other experimental groups were treated with IC-DAN (20 μg mL -1 ) alone named G2, hyperthermia system (IC-DAN + NIR (0.8W cm -2 )) named G4, peroxidase system (IC-DAN + H2O2 (100 μM)) named G3, and their combination (IC-DAN + NIR + H2O2) named G5.The specific experimental operation is as follows.Firstly, mono-colonies of MREC, and MRSA on the solid Luria-Bertani (LB) agar plates were transferred to liquid LB broth and grown at 37°C for 12 h at a rotation shaker of 160 rpm.Then, bacteria were collected in the logarithmic phase of growth.The concentration of bacteria was monitored spectrophotometrically by measuring the optical density at 600 nm (OD600).100 μL of bacteria suspensions (1×10 8 CFU mL -1 ) were treated with five different treatments in a 96-well plate and incubated at 37 °C for 4 h, respectively.100 μL of the bacteria suspension was spread onto LB agar plates and then incubated for 15 h at 37 °C after a series of doubling dilutions.The number of colonies formed units Then, bacteria were dried and sprayed with gold on the surface for further fieldemission SEM images.

Biosafety Assessment of IC-DAN.
Evaluation of Cytotoxicity In Vitro.HDF and HUVEC cells were used for the investigation of the cytocompatibility evaluations.The cells were grown in high glucose DMEM with 1% streptomycin/penicillin and 10% FBS.Cell counting kit-8 (CCK-8) assay was used to measure the cytotoxicity in vitro.The cells were cultured in a 96-well plate (5 × 10 3 cells per well, five wells for each concentration) at a 37 °C humidified incubator with 5% CO2 for 12 h.IC-DAN in different concentrations (0, 25, 50, 100, 150, 200 μg mL -1 ) were added into each well.Then, the cells were coincubated for 24 h and gently washed with PBS three times.According to the manufacturer's protocol, 10 μL CCK-8 diluted into 100 μL DMEM was subsequently added to each well, and the 96-well plate was kept incubated for another 2 h.The absorbance at 450 nm of each well was measured by a microplate reader to evaluate cell viability.Cells treated with PBS were used as control, and the cell viability was calculated as a percentage relative to the control cells.

Red Blood Cells Compatibility Detection. A hemolysis test was used to analyze
the hemocompatibility of IC-DAN in vitro.Briefly, Whole blood cells were collected from healthy people by a 2 mL EDTA-K2 anticoagulant tube.Then red blood cells were separated from whole blood by centrifugation at 2,000 g for 5 min and washed with sterile PBS (pH = 7.4) three times.10% red blood cells were resuspended into PBS and incubated with various concentrations (0, 10, 20, 50, 100, 200 μg mL -1 ) of IC-DAN at room temperature for 2 h.Subsequently, the samples were centrifuged at 2,000 g for 5 min, and the absorbance of the supernatant at 575 nm was measured by a microplate reader.Red blood cells were treated with either PBS or ultra-pure water to be used as a negative (-) or positive control (+), respectively.After an adaptive diet (25 °C, 12 h day/night cycle) for 7 days, a high-fat diet (D12492, 60% calories from fat) for 8 weeks combined with a daily intraperitoneal injection of STZ (40 mg kg -1 day -1 ) for 7 days were used.The fasting blood glucose concentration was measured at more than 11.1 mmol L -1 twice, the mice model was determined to be successful and utilized in the following investigations.Then, the type 2 diabetes mice were randomly divided into five groups (five mice per group).A round wound (d = 8 mm) was cut by surgical scissors on the back of each mouse after anesthesia.Subsequently, MRSA was chosen as the model bacterium for investigating the antibacterial effect of IC-DAN in vivo.The wounds were then infected by 20 μL of MRSA bacterial suspension (1 × 10 8 CFU ml -1 ).After 24 h, IC-DAN (20 μg mL -1 ) was dropped on the wound area with further treatment with H2O2 or NIR in the corresponding groups and PBS solutions in the control group.The wounds of infection were recorded and photographed every three days.

Type 2 Diabetic Mice Wound Infection Model
Hematology and Histological analysis.After 13 days, all mice were sacrificed and the wound tissues were harvested from the mice for further analysis after euthanasia.
Whole blood was collected from mice for routine blood tests.Plasma was separated to measure the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) to analyze the liver function, and blood urea nitrogen (BUN), creatinine (CREA) to assess kidney function, respectively.At the same time, the infected skin tissues in different groups were obtained and fixed with 4wt % formaldehyde solution for hematoxylin and eosin (H&E) staining, Masson staining according to standard operating procedure.The major organs (heart, liver, lung, kidney, and spleen) were excised and fixed with 4 wt % formaldehyde solution for H&E staining.Then the samples were viewed under a digital microscope (Nikon, Japan).
After the experiment, the mice were disposed of following the normal authorized protocol.
Statistical Analysis.OriginPro8.5 was used in the statistical analysis.Number of samples and probability values were showed in figures.All data were shown as means ± standard deviation (SD).Student's t-test was used for two groups and one-way analysis of variance (ANOVA) was used for statistical analysis between multiple groups.
)              Table S3.Comparison of the kinetics for the SAN and IC-DAN toward H2O2.

1 )
and 800 μM TMB and 100 μM H2O2.The absorbance of the solution was monitored at different reaction times for 10 min.By fitting the absorbance data to the Michaelis-Menten equation, we could calculate the catalytic parameters, which present the connection between the substrate and IC-DAN.ESR experiments for hydroxyl radical determination.During the ESR measurements, BMPO was used for hydroxyl radicals scavengers, trapping the radicals and forming adducts that are detectable by ESR spectroscope.In a typical assay, 20 μL of IC-DAN(1000 μg mL -1 ) and 50 μL of H2O2 (100 μM) were added into 100 μL of PBS buffer solution with different pH conditions (pH = 4.0) containing 40 μM of BMPO.The system was then subjected to vortex for 1 min before being transferred to the quartz tube for ESR measurements.The temperature induced ESR measurement was accomplished by coincubating the system in the waterbath at 45℃ for 1 min prior to the measurement.

(
CFUs) were calculated and imaged the next day.The average CFUs numbers were obtained from three duplicate experiments (n = 3).The formula for calculating the antibacterial ratio is C = (A-B)/A × 100%.Where C indicates antibacterial ratio; A is the average CFUs of the control group, and B is the average CFUs of the experimental group.At the same time, according to the staining kit operating instructions, both MREC and MRSA bacteria were stained with SYTO9 and PI for 15 min in the dark after the same treatment.The SYTO9 could stain with all bacteria and PI only enter through damaged bacteria membranes.Thus, it was employed to label all bacteria with green fluorescence and dead bacteria with red fluorescence.The live and dead bacterial cells were visualized with a fluorescence microscope.Morphology Observation of Bacteria by SEM Images.After the antibacterial treatment, five groups of the bacterial suspensions were gently washed and fixed with 2 wt% glutaraldehyde overnight at 4 ℃.After that, the bacteria were dehydrated by sequential treatments with ethanol for 10 min (30, 50, 70, 90, and 100 %), respectively.
In Vivo.To assess the antibacterial effects of IC-DAN in vivo, the diabetic mice wound infection model was built.All animal experiments were authorized by the Institutional Animal Care and Use Committee of Southern Medical University (IACUC Number: SMUL2022216).Firstly, 8-weeks-old Male C57BL/6J mice were employed to generate the type 2 diabetes model.

Figure S6 .
Figure S6.Corresponding EXAFS fitting curves of SAN and IC-DAN at k space

Figure S7 .Figure S9 .
Figure S7.Fe K-edge EXAFS fitting curves of SAN and IC-DAN at R space

Figure S10 .
Figure S10.Wavelet transform of the k 3 -weighted EXAFS data of Fe for SAN, Fe2O3, FePc and Fe foil.

Figure S15 . 2 MRECFigure S16 .
Figure S15.(a) body weight of infected wounds in normal mice and (b) type 2 diabetes mice under different treatments.

Figure S18 .
Figure S18.(A) The relative absorbance of HUVEC and HDF cells after being treated with IC-DAN at different concentrations for 24 h.(B) Hemolysis assessment induced by IC-DAN at various concentrations.Insert picture indicates the direct observation of hemolysis by IC-DAN.

Figure S20 .
Figure S20.Biochemical results of normal infection mice after different treatments.

Figure S21 .
Figure S21.Blood routine test and biochemical results of type 2 diabetes infection mice after different treatments.

S0 2 ,
0.97, is the amplitude reduction factor; N is the coordination number; R is the interatomic distance (the bond length between central atoms and surrounding coordination atoms), σ 2 is Debye-Waller factor (a measure of thermal and static disorder in absorber-scatter distances); ΔE is edge-energy shift (the difference between the zero kinetic energy value of the sample and that of the theoretical model).R factor is used to value the goodness of the fitting.

Table S1 .
XPS and ICP-OES parameters of SAN and IC-DAN.

Table S2 .
Structural parameters of N-CC@Fe SA, N-CC@Fe DA and Fe foil and extracted from the EXAFS fitting.

Table S4 .
Comparison of the kinetics for the SAN and IC-DAN toward TMB.